This study aims to discover the role of Homocysteine-induced ER protein (Herp) deficiency in high-fat diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD). After 8 weeks of feeding with normal-fat diet (NFD) or HFD, WT (wild type) and Herp mice were measured for the body weight, liver weight and serum biochemical parameters. HE, Oil Red O, and Sirius red stainings were used to evaluate the histopathological changes of liver tissues. QRT-PCR, Western blotting and Immunohistochemistry were employed to detect the mRNA and protein expression. TUNEL staining was used to observe the hepatocyte apoptosis. Herp knockout reduced the liver/body weight ratio of mice fed with HFD with the decreased serum levels of TG, TC, HDL, LDL, GGT, Hcy, ALT, and AST. Besides, WT mice fed with HFD presented obvious steatosis, inflammation and hepatocytes ballooning, which was relieved in Herp mice. HFD-induce NFALD mice demonstrated increased Oil Red, Sirius red, and α-SMA staining than NFD-induced mice, but mice in the Herp  + HFD group was lower than the WT + HFD group. HFD-induce NFALD mice showed up-regulated expression of Grp78, Chop, and Atf4 in liver tissues when compared with NFD fed mice. However, regarding to the mice fed with HFD, Herp deficiency decrease in the expression of Grp78, Chop, and Atf4 in liver tissues with the reduced hepatocyte apoptosis. Herp was highly expressed in HFD-induced NAFLD mice. Herp knockout improved liver function and histopathological conditions with the decreased hepatocyte apoptosis and endoplasmic reticulum stress (ERS) of HFD-induce NFALD mice.